PROTEOMIC ANALYSIS OF THE NUCLEOCYTOPLASMIC TRANSPORT MACHINERY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 核孔复合体（NPC）调节真核细胞的蛋白质和RNA在细胞质和核质之间的重要流动。它是一种超分子筛，跨越核膜的两层膜，形成一个40纳米的水通道，连接细胞的细胞质和核质。 小的代谢物通过NPC自由扩散，但是大的蛋白质和RNA的流动是选择性的，并且需要货物中的特定运输信号。 信号被称为核转运蛋白（也称为输入蛋白、输出蛋白和转运蛋白）的移动的受体识别，其与NPC的蛋白质（核孔蛋白; Nups）相互作用以在细胞质和核质之间转运和穿梭货物。 我们正在获得一个蛋白质相互作用的地图之间的54个组件的核质运输机制在S。使用基于GST的亲和捕获测定结合捕获的蛋白质的质谱鉴定来鉴定酿酒酵母。 该方法允许鉴定酵母提取物中的数十种蛋白质，这些蛋白质与运输机械的分离组分（Nups，Kaps，Ran及其效应物）特异性相互作用。 我们目前正在研究如何在不同的细胞条件下调节相互作用网络，例如不同的细胞密度或温度，以及细胞在细胞周期的不同阶段的进展。 该方法提供了核质转运基础的分子动力学的全局图像，以及机器的每个蛋白质组分的许多相互作用的详细地图。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The nuclear pore complex (NPC) regulates the vital flow of proteins and RNA between the cytoplasm and the nucleoplasm of eukaryotic cells. It is a supramolecular sieve that spans both membranes of the nuclear envelope to create a forty-nanometer aqueous channel that connects the cytoplasm and the nucleoplasm of cells. Small metabolites diffuse freely through the NPC, but the flow of large proteins and RNA is selective and requires specific transport signals in the cargo. The signals are recognized by mobile receptors termed karyopherins (also called importins, exportins and transportins), which interact with proteins of the NPC (nucleoporins; Nups) to translocate across and shuttle cargo between the cytoplasm and the nucleoplasm. We are obtaining a map of protein interactions between the 54 components of the nucleocytoplasmic transport machinery in S. cerevisiae using a GST based affinity-capture assay coupled to mass spectrometric identification of captured proteins. The approach has allowed the identification of dozens of proteins in yeast extracts that interact specifically with isolated components of the transport machinery (Nups, Kaps, Ran and its effectors). We are currently examining how the network of interactions is modulated under varying cellular conditions, such as different cell densities or temperature, and as cells progress through different stages of the cell cycle. The approach is providing a global picture of the molecular dynamics underlying nucleocytoplasmic transport, as well as a detailed map of the many interactions that each protein component of the machinery makes.